2. Virus-related safety

Any project related to viruses sounds very dangerous and is therefore in need of an extra biosafety emphasis. However, our group is not working with viruses but instead with Virus-Like Particles (VLPs). VLPs consist of viral Coat Proteins (CPs) which, in the right conditions, will spontaneously assemble into a shell that very much resembles the original virus in shape and size. However, since VLPs consist of only the Coat Proteins, they lack both the external sites that are usually required for the infection of cells and the internal machinery needed for viral replication. Moreover, they lack the viral DNA/RNA to be transcribed and replicated. Consequently, these particles can safely be used without risk of any unintended viral activity. [1,2]
Nevertheless, an overview of which virus components we used and how we got them is included below.

Cowpea Chlorotic Mottle Virus (CCMV)

CCMV is a Risk Group I virus because it poses no threat to humans or animals, but it might nonetheless be harmful for the flora in our direct environment should it ever be accidentally released. However, the CCMV itself or its genome were never in our possession and thus posed no viral threats to our environment.
The viral gene we used encoding the CCMV Coat Protein was obtained from a plasmid stock provided to us by Dr. Kormelink of the faculty of virology at Wageningen UR. The plasmid itself was a standard pET-28a(+) E. coli vector harboring the CCMV Coat Protein encoding sequence (GenBank: NC_003542.1 (1360..1932)) behind an IPTG inducible promotor. No other viral genes were present. This was verified by DNA sequencing.

Hepatitis B Virus (HBV)

HBV is a DNA-based Risk Group III virus because it is considered to pose a high risk for individual researchers. We have no legal classifications nor the experience to work with such a virus. However, the Hepatitis B Virus itself or its genome were never in our possession and thus posed no viral threats to ourselves, other people or the environment.
The gene that we used encoding the Hepatitis B Core Antigen (GenBank: NC_003977.1 (1814..2452)) was obtained from a standard expression vector for in planta expression of proteins (GenBank: GQ497236.1). This plasmid stock was supplied to us, via Dr. Kormelink of Wageningen UR’s Virology faculty, by Hadrien Peyret and George Lomonossoff of the John Innes Centre in Norwich.
No other viral genes were present on this plasmid. This was verified by DNA sequencing. As is the case with other coat proteins, this Hepatitis B Core Antigen protein itself and the gene encoding it are safe for normal laboratory use. [3]

Turnip Yellows Virus (TuYV)

Like CCMV, TuYV is a Risk Group I virus because it poses no threat to humans or animals, but it might nonetheless be harmful to the flora in our direct environment should it ever be accidentally released. The viral gene encoding the Coat Protein for TuYV (GenBank: NC_003743.1 (3483..5495)) was obtained from a plasmid encoding the entire viral genome (GenBank: X13063.1). This plasmid was provided to us, via Dr. Kormelink of Wageningen UR’s Virology faculty, by Véronique Brault of the UMR SVQV in Strasbourg.
With the entire virus genome on one plasmid, it is in theory possible that complete viruses could be formed from it. However, this would require both transcription and translation to take place in significant amounts, which is only possible if a competent cell took it up. This would provide the cell with no foreseeable selective advantage. Still, to prevent this from happening the eppendorf tube containing the plasmids was always handled with Good Microbiological Techniques (GMT) and extra care was taken to keep it sterile. To restrict any risk to a minimum the eppendorf tube containing the plasmid was opened only thrice, to serve as a template for PCR. After completion of our project, this DNA will be destroyed by addition of an excess of hydrochloric acid.
We think that with Good Microbiological Techniques, even in the unlikely case that a virus of TuYV would have formed, the likelihood of it leaving the samples and then the lab, ultimately infecting aphids or plants in our environment, is rather low.

Potato Leafroll Virus (PLRV)

Like CCMV and TuYV, PLRV is a Risk Group I virus because it poses no threat to humans or animals. This virus is native to our direct environment, but accidental release could still mean serious harm to the flora in our environment and in the worst case scenario even an economic disaster for local farmers.
The PLRV genes encoding the viral Coat Proteins were isolated from infected potato plant leaves. The leaves were provided by the ‘Dutch General Inspection Service for Agricultural seed and seed potatoes’ ([http://www.nak.nl NAK]) and sent to us by normal mail service. Part of the leaves was used for RNA isolation, the remainder was frozen immediately at -80°C to minimize the chance of spreading of PLRV. After RNA isolation, cDNA was synthesized using PLRV coat protein specific primers as well as random primers. Subsequent PCR reactions were performed using PLRV coat protein primers. Products of subsequent cloning steps were sequenced and contain only the PLRV coat protein gene. All other (intermediate) products will, after completion of our project, be destroyed.
In nature, PLRV can be spread by several aphid species, most efficiently by Myzus persicae. Since even mechanical transmission (rubbing infected leaves on healthy ones) will not transmit PLRV, spreading of the virus in absence of the insect is almost impossible[4]. 24 Hours after feeding on infected leaves, an aphid may become infectious and transmit the virus to other plants. Both plants and aphids are normally not present in our lab, and the infected plant samples have been treated with Good Microbiological Techniques at all time. Therefore, with reasonable certainty, we can exclude the possibility of this event happening.

References

1. Roy, P. and R. Noad, Virus-like particles as a vaccine delivery system - Myths and facts. Human Vaccines, 2008. 4(1): p. 5-12.

2. Ma, Y., R.J.M. Nolte, and J.J.L.M. Cornelissen, Virus-based nanocarriers for drug delivery. Advanced Drug Delivery Reviews, 2012. 64(9): p. 811-825.

3. Yang, X.-Y., H. Bo, and Y.-L. Shu, Hepatitis B virus core antigen as a carrier for virus-like partical vaccine: a review. Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui], 2012. 28(3): p. 311-6.

4. Mayo, M., et al., Mechanical transmission of Potato leafroll virus. Journal of General Virology, 2000. 81(11): p. 2791-2795.